Our project aims to build the Oxygen hunter, a system that allows for a low oxygen intracellular environment while maintaining normal cellular metabolism, and is expected to provide a "tool" for researchers and companies that require research related to low oxygen intracellular environments. ". We have already contacted a number of companies in the human practice section and received positive responses from them regarding the possible applications we have conceived (bio-hydrogen production, bio-nitrogen fixation, bio-carbon fixation, biological control of water bodies, production of reductive drugs, etc.). (For more information, please click on the implementation page)
In our project, we used leghemoglobin and laccase for intracellular oxygen transport/depletion; in combination with an anaerobically inducible promoter, we used fluorescence intensity to reflect intracellular oxygen concentration (learn more on the design page), thus constructing a detection module and a function module respectively. The data from the experimental results show that the growth of E. coli is only marginally affected, while the intracellular oxygen concentration decreases substantially (result), which serves as our proof of concept.
Please click on the link to see the detailed modelling results (please click model) and human practice feedback (please click integrated human practice) that guided our project.
We first introduced the nirB promoter and laccase or leghemoglobin into different plasmids of E. coli and constructed lacase-function modules (Figure 1-A,B) and leghemoglobin-function modules (Figure 1-C,D), respectively. And performed growth curves and fluorescence intensity measurements. The results showed that the oxygen concentration in E. coli decreased significantly in the presence of the function module (Figure 1-A,C), but its normal growth was only affected to a certain extent (Figure 1-B,D).
Figure 1. Experimental results of single protein building function modules
(A) Laccase
group - different groups of E. coli growth curves (B) Laccase group - different groups of fluorescence
intensity
- time graphs
(C) Leghemoglobin group - growth curves of different groups of E. coli (D)
Leghemoglobin
group - fluorescence intensity of different groups - time line
After iteration, we carried out laccase - leghemoglobin co-expression experiments and obtained the best results in reducing oxygen concentration.
We introduced leghemoglobin, laccase and nirB promoter into plasmids of E. coli and successfully expressed them (Figure 2-A,B,C) and performed growth curve (Figure 2-D) and fluorescence intensity (Figure 2-E) measurements.
Figure 2. Results of modeling simulations of laccase - leghemoglobin co-expression
experiments
(A) Schematic diagram of the working mechanism of the system (B) PCR results of nirB promoter
(C) SDS results of different groups (D) E. coli growth curves measured by different groups
(E)
Fluorescence intensity-time plots of different groups
The results of the experiment show that, on the one hand, the fluorescence intensity of the E. coli introduced with the function module increased significantly compared to the blank group with only nirB introduced (Figure 2-E), which indicates that the intracellular oxygen concentration decreased significantly and the functional module played a role; at the same time, combined with the growth curve, it can be seen that the normal growth of the E. coli introduced with the function module was affected to a certain extent compared to the control group with only the nirB promoter introduced (Figure 2-D). This is due to the fact that the expression of exogenous proteins increases the metabolic burden on the cells. While,, the graph also shows that the E. coli colony numbers have remained largely stable and thus we have successfully carried out a proof of concept for the project.
Overall, the above experimental results show that our Oxygen Hunter system can achieve the construction of an intracellular hypoxic environment while basically maintaining normal cell metabolism. It can be used in many fields such as biological nitrogen fixation, biological hydrogen production, biological carbon sequestration, synthesis of reducing drugs and biological treatment of water bodies, which has good prospects.